Vesicular Traffic, Secretion, and Endocytosis Part 1

Lecture 14

transport vesicles, endoplasmic reticulum, golgi complex, nuclear envelope, endoscopes, and lysosomes are all part of the

endomembrane system

secretory and endocytic pathway protein trafficking - unifying principle:

transport vesicles transport membrane and soluble proteins from one membrane-bounded compartment to another 

transport vesicles collect

cargo proteins in membrane budding from a donor compartment

transport vesicles deliver

cargo proteins to the next compartment by fusion with the target membrane

secretory pathway - distribution of

soluble and membrane proteins synthesizes by the rough ER to final destinations at the cell surface (including secretion) or in lysosomes

secretory pathway : stage 1

rough endoplasmic reticulum

secretory pathway : stage 2

protein trafficking

secretory pathway : stage 1 stage 1 synthesis of protien bearing an ER signal/targeting sequence -

cotranslational insertion of newly made polypeptide chains into the ER membrane or across it into the ER lumen 

secretory pathway : stage 2 step 2

proteins packaged into transport vesicles that bud from the ER and fuse together to form a new cis-Golgi cisternae 

secretory pathway : stage 2 step 3 ER enzymes or structural proteins -

• retained in the ER 

  • retrieved to the ER by vesicles that bud from the cis-Golgi and fuse with the ER

secretory pathway : stage 2 step 4

each cis-Golgi cisterna and contents moves from the cis to the trans face of the Golgi complex by nonvesicular cisternal maturation 

secretory pathway : stage 2 step 5

retrograde transport vesicles move Golgi-resident proteins to the previous Golgi compartment 

secretory pathway : stage 2 step 6 constitutive decoration (all cells) - transport vesicles move continuously and fuse with the plasma membrane 

• soluble proteins are continuously secreted

• membrane proteins become plasma membrane proteins 

secretory pathway : stage 2 step 7 regulated secretion (certain cell types)

• proteins accumulated and stored in regulated secretory vesicles

• vesicles fuse with plasma membrane and secrete proteins only when cell recieves a neuronal or hormonal signal secretion signal 

secretory pathway : stage 2 step 8 lysosome-destined membrane and souble proteins -

transported in vesicles that bud from the trans-Golgi and fuse with the late endosome for delivery to a lysosome 

endocytic pathway: step 9 

vesicles budding from the plasma membrane take up soluble extracellular proteins and deliver them to lysosome via late endosomes 

three types of coated vesicles mediate

proteins transport through different pathways

small GTPase proteins direct

coat protein polymerization on donor membranes to pinch off vesicles carrying different cargoes 

coat shedding exposes

Rab and SNARE proteins that target vesicles for fusion with specific target membranes 

GTPase superfamily proteins exist in two forms/conformations

GTP-bound and GDP-bound

GTPases hydrolyze

GTP to GDP

GTP-bound form:

active “on” conformation - can interact with target proteins to regulate their activities 

GDP-bound form:

inactive “off” conformation

GEF (guanine nucleotide exchange factor) :

stimulates replacement (exchange) of the bound GDP (off) with a GTP (on)

GAP (GTPase-activiating protein)

stimulates GTP (on) hydrolysis to GDP (off)

vesicles 

• bud from a donor membrane

• fuse with specific target membrane

assembly of a protein coat drives

vesicle formation and selection of specific cargo molecules

recruitment of GTP-binding G proteins to

a region of donor membrane

cytosolic coat proteins complexes bind to

cytosolic domain of membrane cargo proteins

coat binding evaginates

the membrane

some cargo proteins acts as receptors to 

bind soluble proteins in the lumen and capture them into the budding vesicle 

donor membrane-specific SNARE proteins captured in 

budding vesicle membrane

donor membrane fusion

pinches off coated vesicle 

coated vesicle uncoated in cytosol 

uncovers projecting v-SNARES for fusion targeting 

vesicle fusion targeting

interaction of specific v-SNARE with specific target membrane t-SNARES

three major types of transport-specific coated vesicles

each with a different type of protein coat formed by reversible polymerization of distinct set of coat and G protein subunits

A conserved set of monomeric GTPase switch proteins control

the assembly of different vesicle coats

COPII-coated vesicles -

move materials from the ER to the Golgi complex (Anterograde Movement)

COPI-coated vesicles

move material from Golgi “backward” to ER, or form the trans Golgi to the cis Golgi cisternae (Retrograde Movement) 

Clathrin-coated vesicles

move materials from the TGN to endosomes, lysosomes, and plant vacuoles

cycle of GEF-activated GTP binding and GAP-activated GTP-hydrolysis controls

assembly and disassembly of vesicle coats 

mechanisms are well conserved between the Sar1 GTPase (COPII) and ARF GTPase (COPI) and clathrin:

1. Sar1 membrane binding, GTP exchange

2. COPII coat assembly

3. GTP hydrolysis

4. coat disassembly

COPII vesicle recruitment of cargo proteins: Sar1-GTP

recruits Sec23/Sec24 to ER membrane

COPII vesicle recruitment of cargo proteins: Sec24 interaction with cytosolic domain di-acidic targeting signal recruits

cargo protein into the vesicle membrane

COPII vesicle recruitment of cargo proteins: soluble cargo proteins interact 

with luminal regions of membrane cargo proteins 

Rab GTPases control

docking of vesicles on target membranes 

fusion of secretory vesicles with the plasma membrane -

similar mechanism mediates all vesicle-fusion events

v-SNARE and t-SNARE complex

four long alpha helices, two from SNAP-25 and one each from synthaxin (t-SNARE) and VAMP (v-SNARE), form numerous noncovalent interactions to form four-helix coiled-coil

formation of four-helix bundle is

energetically favorable and can overcome the electrostatic repulsion of the phospholipid heads 

formation of four-helix bundle allows to hydrophobic interiors to 

come into contact and create and opening between the two membranes

• the membranes fuse together and hydrophobic interactions reorder the phospholipids into a bilayer

COPII-coated vesicles transport

newly synthesizes protein containing Golgi-targeting sequence in their cytosolic domain or bound to such proteins from the rough ER to the cis0Golgi (anterograde direction) 

COPI-coated vesicles transport

vesicles carrying ER/Golgi-resident proteins in the retrograde direction, which supports Golgi cisternal maturation 

Golgi complex is a

stack of flattened membrane cisternae, some cisterna continuous with others 

Golgi complex receives COP coated vesicles from

the ER and further modifies protein in transit and sorts them for delivery 

many golgi per cell, number depends of

degree of protein production

cis Golgi Network (CGN)

• entry site for material arriving from ER

  • some proteins separated, packages into vesicles and recycles to ER

  • other proteins sent on to other regions of Golgi 

proteins are maintained in an organelle by

a combination of two mechanisms

mechanism 1 of proteins maintained in an organelle: retention of resident molecules that are

excluded from transparent vesicles 

mechanism 2 of proteins maintained in an organelle: retrieval of “escaped”

molecules back to compartment in which they reside

proteins that normally reside in ER contain

short amino acid sequences at their C-terminus that serve as retrieval signals 

specific receptors capture

the molecules and return them to ER in COPI coated vesicles

KDEL receptor:

recognizes and returns soluble ER proteins based on Lys-Asp-Glu-Leu (KDEL) signal 

ER membrane proteins have a

KKXX retrieval signal on their C-terminus which binds to COPI coat

vesicle transport between ER and cis-Golgi -

initial transport stage of secretory pathway

formward (anterograde) transport -

COPII vesicle-mediated ER to cis-Golgi transport

cargo-

newly synthesized proteins

reverse (retrograde) transport - 

COPI vesicle-mediated cis-Golgi to ER transport 

cargo-

recycles the membrane bilayer, v-SNAREs, and missorted ER-resident proteins

cis-Golgi network -

ER-to-Golgi intermediate sorting compartment

soluble ER luminal resident proteins:

• function in the ER to modify newly synthesized proteins 

• several types contain a C-terminal KDEL (Lys-Asp-Glu-Leu) ER-targeting sequence

cis-Golgi network -

• membrane contains KDEL receptors 

• acidic pH - promotes receptor-KDEL interaction

retrieved system prevents 

depletion of ER luminal proteins needed for proper folding of newly made secretory proteins 

addition of KDEl sequence to C-terminus of protein normally

secreted - becomes localized in ER 

polarity to Golgi: cis face of Golgi closely to ER

network of tubules = cis Golgi network (CGN)

polarity to Golgi: trans face of Golgi away from ER

network of tubules = trans Golgi network (TGN)

cisterna formed by fusion of

vesicles at cis face

• moves to next position as new cisterna formed 

  • progresses down Golgi stack

discrete enzymatic activity in each region of the Golgi and there enzymes are moved in an 

anterograde fashion along with the proteins being processed 

retrograde movement of transport vesicles (COP coated) returns these

enzymes to their appropriate positions both in the Golgi and ER

evidence for cisternal maturation model - block ER transport vesicles leads to 

disappearance of Golgi 

evidence for cisternal maturation model - materials produced in ER retina in the golgi complex and

never appear within the Golgi-associated transport vesicles 

evidence for cisternal maturation model

• data suggests that vesicles can move forward (anterograde) or backward (retrograde) 

• composition of individual Golgi cisterna change over time 

movement of material 

CGN → cis cisterna → medial cisternas → trans cisterna → TGN

membraneous element of Golgi complex supported mechanically by

a peripheral membrane scaffold 

scaffold physically linked to

motor proteins that direct movement of vesicles

golgi “matrix” is a group of

fibrous proteins that play a key role in the disassembly and reassembly of the Golgi complex during cell division

golgi complex - three biochemical processing compartments contain

different enzymes that modify proteins post-translationally 

anterograde transport through the three golgi processing compartments occurs 

by cisternal maturation 

glycosylation in the golgi complex

• sequence of incorporation of sugars into oligosaccharides is determined by glycosyltransferases in each region of the golgi 

• glycoslyation steps can be diverse 

other function associated with golgi complex - synthesis and modification

• sphingomyelin synthesis completed 

• O-linked oligosaccharides added to proteins

  • attached to oxygen of serine or thyronine 

other function associated with golgi complex -site of synthesis of most cell’s complex polysaccharides 

• Polysaccharides of extracellular matrix of animal cells

• Polysaccharides of cell walls of plants except cellulose

other function associated with golgi complex -

addition of oligosaccharides to lipids 

the trans-golgi networks sorts

proteins into vesicles targeted for different destinations

lysosomal enzymes bear

M6P residues that are recognized by M6P-receptors and delivered by a clathrin-coated vesicles pathways to lysosomes

regulated secretory proteins are concentrated and stored until

secretion is signals; constitutively secreted portions are continuously delivered to the plasma membranes

ome proteins are processes into mature form after

leaving the trans-golgi network